Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.


Introduction
Oxidative stress has been linked to many disorders including neurological pathologies.
Recently, the term has been redefined and divided into oxidative eu-and distress, acknowledging redox regulation of specific targets in physiological signal transduction [1,2].
Oxidative distress can induce neuronal cell death and is widely considered as a pivotal cause of cell death in neurodegenerative disorders, such as Alzheimer's (AD) or Parkinson's disease (PD) [3,4]. In the last decades, pathophysiological mechanisms contributing to neuronal cell damage through different forms of regulated cell death (RCD) were studied intensively [5]. It is widely accepted that major steps of the cell death cascade comprise detrimental accumulation of intracellular calcium and formation of reactive oxygen species (ROS) [3,6].
Moreover, different RCD paradigms converge at the level of mitochondria [7]. Mitochondria are dynamic organelles regulating the energy metabolism, calcium homeostasis and the cellular redox balance [8]. Thus, mitochondrial demise, including mitochondrial calcium overload, loss of the mitochondrial membrane potential, accumulation of reactive oxygen species and release of apoptosis inducing factor (AIF) are considered as the 'point of no return' upon cell death induction [9,10]. A broad understanding of the molecular mechanism involved in transducing detrimental cell death signals to mitochondria are of great importance for future clinical implications.
In the current study, regulated cell death was induced by glutamate or erastin treatment leading to cell death mechanisms called oxytosis or ferroptosis, respectively. Oxytosis is a well-established form of regulated cell death occurring during neuronal development, as well as under pathological conditions in neurodegenerative diseases [11]. In addition, ferroptosis was defined more recently as an iron-dependent form of oxidative cell death, which can be achieved, e.g., by erastin treatment in neuronal HT22 cells [12,13]. Both forms of cell death share very similar RCD mechanisms in neuronal cells, and both, glutamate-or erastin-induced inhibition of the cystine-glutamate (XC -)-antiporter leads to a reduction in glutathione levels by depletion of intracellular cysteine. This results in impaired activity of the glutathione peroxidase-4 (Gpx4) and activation of 12/15-lipoxygenase (LOX) and accumulation of ROS [14,15]. In turn, dynamin-related protein 1 (DRP1) and the pro-apoptotic protein BID attain activity to translocate to mitochondria inducing mitochondrial ROS production and loss of the mitochondrial membrane potential by mitochondrial outer membrane permeabilization (MOMP) [13,16,17]. Finally, cytochrome c and apoptosis inducing factor (AIF) are released from mitochondria and translocate to the nucleus, where AIF is involved in the degradation of deoxyribonucleic acid (DNA) [14,15,18].
Cofilin1 is a member of the ADF/cofilin family of actin-depolymerizing proteins and the major representative of this family in neurons [19]. Upon dephosphorylation of serine residue at position 3 (Ser3) of the protein, it can bind to filamentous actin (F-actin) and initiate its depolymerization [20]. Moreover, it can bind to globular actin monomers (G-actin) and inhibit the nucleotide exchange from ADP-actin to ATP-actin, which is required for F-actin assembly [21]. Thus, cofilin1 can exert indirect effects on molecular mechanisms by operating on actin dynamics and it can also act as a direct participator in the apoptotic cell death cascade by recruitment of cofilin1 from the cytosol to mitochondria [22]. Importantly, cofilin1's effects on mitochondria can be versatile, as it was shown to be involved in transducing apoptotic signaling to mitochondria upon oxidation [22,23]. Oxidation of cofilin1 is an important posttranslational modification for the regulation of cytoskeletal dynamics (reviewed in [24]). Moreover, acting on mitochondrial dynamics via DRP1 activation was demonstrated in a cofilin1 loss-of-function approach in mouse fibroblasts [25].
How cofilin1 can also participate in non-apoptotic cell death paradigms remains to be elucidated. Therefore, we investigated the effect of cofilin1-depletion in non-apoptotic cell death induced by glutamate or erastin in neuronal HT22 cells and primary cortical neurons.
The results obtained in this study demonstrate a role for cofilin1 as a redox sensor and regulator of oxytosis or ferroptosis, upstream of mitochondria, in neuronal cells.
For efficient cofilin 1 knockdown, HT22 cells were transfected with 15 nM cofilin1 siRNA for 48 h using Lipofectamine RNAiMAX (Thermo Fisher Scientific, Darmstadt, Germany) according to the manufacturer. Control cell were transfected with unspecific scrambled control siRNA. Following siRNA sequences were obtained from Dharmacon: scrambled siRNA

Primary cortical neurons
Primary cortical neurons were prepared from embryonic mouse brains (E18) as described previously [18]. Dissociated neurons were seeded at a density of 45

Cofilin1 flx/flx mice
Genetically modified mice expressing Cofilin1 allele with exon 2 flanked by loxP sites were used as controls (Ctrl) [26]. Cofilin1 knockout was achieved by expression of the Cre enzyme capable of recognizing loxP sites and thus specifically deleting exon 2 of the cofilin1 gene region, resulting in a non-functional gene product. Since a systemic knockout of cofilin1 is embryonically lethal [27], Cre expression is under the control of a CaMKIIα-promotor to specifically delete cofilin1 in excitatory neurons for the forebrain including cerebral cortex neurons [28].

Protein analysis
Protein extraction and Western blot analysis were performed as previously described [13]. Analysis (RTCA; Roche Diagnostics, Mannheim, Germany) system as previously described [29]. Changes in the impedance are depicted as normalized cell index. HT22 cells were seeded in 96-well plates at a density of 6,000-8,000 cells per well.

ATP measurement
Cellular ATP levels were measured using the ViaLight Plus Kit (Lonza, Verviers, Belgium) according to the manufacturer's protocol. Briefly, cells were lysed, transferred to a white-walled 96-well plate and the ATP monitoring reagent was added to the cell lysate. Afterwards, the luminescence was detected with a FLUOstar OPTIMA reader (BMG Labtech, Ortenberg, Germany).

Measurement of mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate
Determination of the mitochondrial oxygen consumption rate as an indicator of mitochondrial respiration was performed using the Seahorse XFe96 Analyzer (Agilent Technologies, Waldbronn, Germany). Cells were plated in XFe96-well microplates (6000 cells/well, Seahorse Bioscience) and 1 h prior to the measurement, growth medium was replaced by the seahorse assay medium (4.5 g/l glucose, 2 mM glutamine, 1 mM pyruvate, pH 7.35). After recording three baseline measurements, four compounds were added by injection.

Mitochondrial isolation
Mitochondrial isolation of freshly dissected cortical or hippocampal brain tissue (~50 mg) was performed as previously described [18].

Expression, purification and thermal stability
The human cofilin1 WT, the mutant lacking two Cys residues (Cys139/147Ser, 2Cys → Ser) and the mutant lacking all 4 Cys residues (4Cys → Ser) were expressed as His-Tag fusion proteins in E. coli as described before [30]. The proteins were purified by immobilized metal affinity chromatography using the His Trap Kit from GE Healthcare Life Science, USA. Expression and purification efficiency were analyzed by SDS-PAGE using precast gels from BioRad, USA, and Coomassie staining. Proteins were re-buffered into PBS using Zeba Spin columns and the thermal stability of the proteins was analyzed by recording the emission at 600 nm over time with increasing temperature from 20 °C to 70 °C (2 °C per 3 min) using the Shimadzu UV1800.
Recombinant cofilin1 was either used in the native way, oxidized by 100 µM H2O2 incubation for 30 min, or reduced with 10 mM freshly dissolved dithiothreitol (DTT) for 30 min. The remaining elution buffer from the protein purification process was substituted by PBS using sephadex-based PD MidiTrap G-25 columns (GE Healthcare, Chicago, USA). Afterwards, protein amount was determined by a NanoPhotometer™ (Implen, Munich, Germany). The experiments were performed using 0.13-0.25 µg recombinant protein per µg mitochondrial protein and incubated for 30 -60 minutes at room temperature and another 10 minutes at 37 °C and afterwards measured as indicated at the respective method.

Cofilin1 downregulation attenuates glutamate-and erastin-induced cell death
The phosphorylation and oxidation state of cofilin1 determines not only its binding capacity to F-actin [31][32][33], it is also essential for translocation to mitochondria [23]. In particular, dephosphorylated cofilin1 attains activity for translocation from the cytosol to mitochondria [34]. Here, mmortalized mouse hippocampal HT22 cells were used to examine the relevance of cofilin1 involvement in neuronal cell death mechanisms.
Cofilin1 involvement in cell death pathways, such as glutamate-induced oxytosis or erastininduced ferroptosis in neuronal HT22 cells and its dependency on the cofilin1 phosphorylation status was demonstrated by Western blot analysis. Cofilin1 was dephosphorylated between 8 and 14 h after the administration of glutamate or erastin, respectively ( Fig. 1 A, B). Cofilin1 protein levels started to diminish after 16 h of damaging the cells with erastin or glutamate (Fig.   1A, B). In cell death paradigms with oxidative distress as a prerequisite, a loss-of-function approach was valuable to specify the importance of cofilin1. Therefore, cofilin1 downregulation was achieved by incubation with two different siRNA sequences (si01, si02) for 48 h. The knockdown efficacy at mRNA level was detected by RT-PCR with specific primers leading to an amplification product with a size of 146 base pairs (Fig. 2 B). Gapdh primers were used as a loading control. At the protein level, Western blot analysis revealed an efficient knockdown of cofilin1 (Fig 2 A). At the molecular level, knockdown of cofilin1 was sufficient to prevent cellular damage after 16 hours of erastin or glutamate exposure. Particularly, the metabolic activity, representing the viability and survival of cells, revealed a protective outcome of cofilin1-knockdown upon erastin or glutamate treatment (Fig. 2 C), as assessed by MTT assay [35].

Cofilin1 acts downstream of lipid peroxidation and cellular reactive oxygen species formation, but upstream of mitochondrial demise
Oxidative distress is considered a major hallmark in oxytosis and ferroptosis [36,37].
Peroxidation of lipids represents a hallmark in cell death cascades comprising reactive oxygen species downstream of glutathione depletion in models of oxytosis and ferroptosis [13,15,38].
To further validate the specific activity of cofilin1, lipid peroxidation was assessed using the fluorescent dye BODIPY C11 and flow cytometry; ROS were assessed using H2-DCF. Of note, both, erastin and glutamate treatment induced pronounced accumulation of lipid peroxides, which was not affected by siRNA-mediated cofilin1 knockdown ( Fig. 3 A), indicating that lipid peroxidation occurs upstream of detrimental cofilin1 activation. Interestingly, cofilin1 depletion could not diminish the fluorescent DCF signal (Fig. 3 B), thus, in the applied model systems of oxidative cell death, cofilin1 activation occurs downstream of cellular ROS generation. Next, we used the MitoSOX marker that specifically detects mitochondrial superoxide. Mitochondrial involvement is widely considered as the 'point of no return' in cell death paradigms induced by millimolar doses of glutamate [10,39,40]. However, the relevance of mitochondrial damage in ferroptosis induction is controversially discussed [12,13,41,42] and may depend on the experimental settings and, in particular, on the cell type. In this regard, it was essential to investigate possible links between ferroptotic signaling and mitochondrial demise. Intriguingly, both, glutamate and erastin challenge triggered massive mitochondrial superoxide accumulation, however, cofilin1 depletion completely blocked such mitochondrial superoxide formation (Fig. 3 C). To address the impact of cofilin1-knockdown on the mitochondrial membrane potential, TMRE staining and flow cytometry analysis were conducted. After glutamate or erastin exposure, the membrane potential was considerably impaired, and this was attenuated by cofilin1 knockdown (Fig. 3 D), thereby preserving mitochondrial integrity, represented by the mitochondrial membrane potential, which is crucial for proper energy storage upon oxidative phosphorylation processes [43]. Cofilin1 depletion without any further treatment neither impaired mitochondrial ROS generation, nor the mitochondrial membrane potential. Further, glutamate and erastin treatments resulted in an overall decline of ATP levels, measured by a luminescence-based approach. Depletion of cofilin1 was capable of partly rescuing the effect on ATP production indicating that, to a certain extent, the energy supply was preserved (Fig. 3 E). Massive mitochondrial calcium accumulation is considered as a detrimental prerequisite for mitochondrial impairment, eventually provoked by increased calcium-induced mitochondrial respiration, nitric oxide production and finally, loss of mitochondrial membrane integrity [44]. To address mitochondrial calcium alterations, Rhod-2 AM staining and flow cytometry measurements were performed. The results revealed, that cofilin1 depletion could significantly reduce the massive mitochondrial calcium accumulation following glutamate or erastin exposure (Fig. 3 F).
With regard to energy metabolism, the Seahorse XFe96 Analyzer was used to determine both mitochondrial respiration by measuring the oxygen consumption rate (OCR) and glycolysis by measuring the extracellular acidification rate (ECAR). This measurement revealed complete inhibition of mitochondrial respiration and a diminished glycolysis rate after glutamate or erastin treatment in neural HT22 cells (Fig. 4 A-D (Fig. 4 B, D), suggesting a metabolic switch towards glycolytic-based energy production in cofilin1-deficient neurons exposed to oxidative stress. In this regard, correlation between OCR and ECAR illustrates the metabolic potential of the cells, measured under baseline and stressed conditions by FCCP injection (Fig. 4 E, F). Especially after glutamate or erastin treatment, metabolic bioenergetics underwent a mostly quiescent state in control conditions, whereas cells deficient of cofilin1 exhibited a considerably higher metabolic potential, indicating a functional energy production during oxidative stress (Fig. 4 E, F). Of note, under control conditions, cofilin1 knockdown itself significantly shifted the metabolic state led to a significant reduction of the metabolic activity, which was prevented by the NMDAreceptor antagonist MK801 (Fig. 5 B). In cofilin1-deficient neurons, the detrimental impact of glutamate was entirely abrogated, similar to the effects of MK801 (Fig. 5 B). Overall, excitotoxicity exerts detrimental effects on mitochondrial integrity and function, including deficits of ATP production as a consequence of impaired mitochondrial respiration. To gain further insight into the metabolic activity of primary neurons, we performed seahorse measurements. These assays revealed that the oxygen consumption rate, as an indicator of mitochondrial function, was decreased after glutamate exposure of Ctrl neurons under basal conditions and upon evoking maximal respiration by FCCP (Fig. 5 C, E, F). MK801 was able to prevent the detrimental impact of glutamate on basal and maximal OCR (Fig. 5 C, E, F).
Similarly, mitochondria of cofilin1 knockout neurons were also significantly protected against glutamate-induced loss of basal and maximal respiration (Fig. 5 D, E, F), indicating that cofilin1 mediated mitochondrial damage in models of glutamate excitotoxicity in the cortical neurons.
As mentioned before, the phosphorylation status of cofilin1 Ser3 is considered to be a phosphorylation, Western blot was performed using a specific antibody against phosphorylated Ser3-cofilin1. As clearly demonstrated, cofilin1 was dephosphorylated after glutamate exposure, whereas CN03 preserved the phosphorylation status (Fig. 6 A). The effect of this manipulation was assessed in the MTT assay to quantify metabolically active cells.
Interestingly, a 3-hour pretreatment with 1 µg/mL CN03 rescued the loss of metabolic activity induced by 24-hour exposure of glutamate (Fig. 6 B). This beneficial effect was comparable to the potent NMDA-receptor antagonist MK801 (Fig. 6 B).

Administration of recombinant cofilin1 on isolated mitochondria impaired the mitochondrial membrane potential and respiration
Besides the well-established function on F-actin dynamics, cofilin1 has been linked to oxidative cell death, e.g. induced by the oxidant taurine chloramine [22] or H2O2 [46,47].
Cofilin1 possesses several cysteine residues essential for the quaternary structure of the protein by forming intra-or intermolecular disulfide bonds. In human cofilin1, four crucial cysteines have been described prone to oxidation at positions 39, 80, 139 and 147 [24].
Dephosphorylation of Ser3 and oxidation of the aforementioned cysteine residues are considered as crucial prerequisites for mitochondrial localization after apoptosis induction [22].
To specifically address the impact of WT cofilin and specific mutants (2Cys: Cys139Ser/Cys147Ser; 4Cys: Cys39Ser, Cys80Ser, Cys139Ser, Cys147Ser), we cloned different constructs, expressed the proteins in E. coli and purified it using the IMAC principle.
Proteins were re-buffered into PBS and the impact of Cys mutations on the thermal stability was analyzed at 600 nm using a UV-spectrophotometer. The 2CysSer mutant showed a similar stability as the WT protein ( Supplementary Fig. 1). The 4CysSer mutant was less stable than the other proteins. However, all recombinant proteins were stable at the temperature of 37 °C that was used for all experiments on isolated mitochondria ( Supplementary Fig. 1). The impact of recombinant cofilin1 was analyzed on mitochondrial superoxide formation, mitochondrial membrane potential and mitochondrial respiration. Mitochondria isolated from mouse brain were incubated with or without the respective cofilin1 variants under basal, oxidized (H2O2) or reduced conditions (DTT). Strikingly, the reduced form of WT cofilin1 had no impact on the membrane potential, whereas application of WT cofilin1 either in the natural form or in the oxidized state decreased the mitochondrial membrane potential. Further, the Cys139/147Ser mutation as well as conversion of all four cysteines to serine completely abolished the effect of cofilin1 on isolated mitochondria (Fig. 7 A). The chemical ionophore CCCP was used to demonstrate the maximal effects of mitochondrial membrane collapse measured by TMRE staining (Fig. 7 A).
To further elucidate the impact of the WT protein and the 2Cys-cofilin1 mutant on isolated mitochondria, mitochondrial superoxide generation was measured using MitoSOX staining.
The maximal effect of mitochondrial superoxide generation was evoked by antimycin A treatment, a potent complex III-inhibitor of the respiratory chain (Fig. 7 B). The oxidized form of the WT protein also induced a significant burst of mitochondrial superoxides, whereas the reduced WT protein and the 2Cys-mutant generated comparable ROS levels to mitochondria of the untreated control group (Fig. 7 B). The effect of the serine mutants on mitochondrial integrity and superoxide generation was therefore mainly attributed to the cysteine residues at position 139 and 147.
Finally, to understand the effect of recombinant cofilin1 at a functional level, mitochondrial bioenergetics were evaluated using the Seahorse XFe Analyzer. The mitochondrial assay buffer (MAS) contained succinate to specifically assess complex II-driven respiration and rotenone to prevent reverse electron flow. ADP injection allows for specific calculation of the phosphorylating capacity to produce ATP. The analysis of the ADP-driven mitochondrial activity revealed a significant impairment (p<0.02) of the measured OCR of mitochondria challenged with the oxidized WT cofilin1 protein compared to the control condition (Fig. 7 C, D, E). The oxidized form of the 2Cys mutant had a slightly minor derogating impact on the ADP-dependent respiration compared to the WT cofilin1 protein (WT p<0.02 vs. 2Cys p<0.04), which was completely reversed by reduction of these cysteine residues (Fig. 7 D, E, F).
Oligomycin, a potent ATP synthase inhibitor (complex V), allowed for estimating the proton leak across the inner mitochondrial membrane, which was not apparently affected after application of either the oxidized or reduced form of the recombinant protein ( Fig. 7 C, D).
Injection of the uncoupler FCCP disrupts the proton gradient to facilitate maximal respiration.
Again, this state of mitochondrial respiration was not compromised by either the WT cofilin1 or the 2Cys cofilin1 mutant (Fig. 7 F).

Discussion
The present study identified a pivotal role of cofilin1 upstream of mitochondrial damage in oxidative cell death induced by glutamate or erastin and in models of glutamate excitotoxicity in primary cortical neurons. Here, we demonstrate that cofilin1 downregulation in neuronal HT22 cells by specific cofilin1-targeting siRNA or by genetic deletion in primary neurons, exerts protective effects on mitochondrial function and cellular resilience. We have shown that cofilin1 deficiency affects mitochondrial superoxide accumulation, mitochondrial membrane potential, mitochondrial calcium accumulation, mitochondrial respiration, and ATP generation.
Mitochondrial superoxide production by complex I and III occurs during respiration.
Moreover, it can either be a result of complex I defects, genetic abnormalities or excessive mitochondrial calcium admission [48][49][50]. Massive mitochondrial calcium gathering induces the opening of the mitochondrial permeability transition pore and subsequent loss of the mitochondrial membrane potential leading to cell death (reviewed in [51]). Our results emphasize that cofilin1 depletion can reduce massive mitochondrial calcium overload which may account for the protective mechanism upon cofilin1 depletion. In this regard, possible indirect effects by alteration of the actin cytoskeleton might be conceivable to impact mitochondrial calcium uptake, as previously discussed for INF2-knockout cells [52].
Mitochondrial ROS is especially detrimental for mitochondrial function, as it leads to mitochondrial DNA damage and subsequent impaired oxidative phosphorylation (OXPHOS) [53][54][55]. Therefore, preventing mitochondrial ROS accumulation by cofilin1 depletion is an Excessive glutamate accumulation is a hallmark of several neurodegenerative diseases, stroke and brain trauma [57,58]. Many hallmarks of neuronal loss are already identified to facilitate the development of possible treatment strategies for these diseases. However, how different cell death mechanisms are involved and at which point they converge is not completely understood. Cofilin1 was identified as a key player in different neurological diseases, e.g. in Alzheimer's or Parkinson's disease [59][60][61][62]. Recent findings also imply a role for cofilin1 in ischemic brain damage [63,64]. In this paradigm, cofilin1 phosphorylation was able to prevent detrimental cofilin-actin rod formation, thereby improving mitochondrial transport failure induced by oxygen and glucose deprivation [63]. In our study, CN03-induced phosphorylation also impressively protected neurons from glutamate-induced cell death revealing a potential target for future treatment strategies, conceivable for a variety of neurological disorders, such as stroke [63] or autism-like deficits [65]. A putative mechanism involves Rho activation by CN03, thereby phosphorylating cofilin1 via ROCK-LIMK pathways and finally promoting neuronal protection by circumventing cofilin1 activation and mitochondrial demise (Fig. 6 C).
Underlining the strong effect of our loss-of-function-approach, evidence exists, showing a direct effect of cofilin1 by binding to mitochondria [22,66,67]. In this regard, it was demonstrated that cofilin1 gains activity to translocate to mitochondria upon oxidation by taurine chloramine, a physiological oxidant derived from neutrophils [22,34]. In line with these results, our data suggest that cofilin1 is an important mediator upon oxidative distress, as cofilin1 depletion was sufficient to block mitochondrial damage and, thereby, the oxidative cell death cascades. In order to distinguish potential indirect actin-based cofilin1 effects from those which are directly mediated by cofilin1, isolated mitochondria were incubated with recombinant cofilin1 protein. The functional measurements revealed a direct detrimental effect of cofilin1 on mitochondrial integrity and respiration. First hints of direct cofilin1-mediated impacts on mitochondria were given by Chua and coworkers in 2003 [23]. A few years later, four cysteine (39/80/139/147) and three methionine residues were identified, which are prone to oxidation, but only the oxidized cysteines were linked to mitochondrial demise [22]. In particular, a detrimental role for the oxidized form of cofilin1 upstream of mitochondria was unraveled in cell death models induced by the oxidants H2O2 or taurine chloramine (TnCl) [22,68]. Under these conditions, cofilin1 can translocate to mitochondria and induce mitochondrial swelling, cytochrome c release and open the mitochondrial permeability transition pore (mPTP). This mitochondrial transactivation of the protein was even observed under basal conditions without any further stimulus when the cells express the oxidation-mimetic glycine residues at position 39 or 80, respectively [68]. Apparently, cysteines do not only serve as redox sensors and mediate redox signaling, they are also crucial for the correct structural protein formation and interaction. Especially Cys39 and Cys80 were described to form intramolecular disulfide bonds and their oxidation eventually led to protein dephosphorylation (Ser3) after oxidation due to sterical effects [24]. Cys139 and Cys147 are able to form both, intra-and intermolecular disulfide bonds, thus presenting a prerequisite for cofilin1 oligomerization [69]. In the present study, specific mutations of either two (Cys139/147) or all four cysteine residues of the recombinant protein were realized to address the question which specific cysteine residues contribute to the deleterious effects of the protein after oxidation. Specific evaluation of mitochondrial parameters after incubation of isolated mitochondria with cofilin1 facilitated insights into the direct mechanism of cofilin1 without any other cellular components.
Intriguingly, the wildtype form of cofilin1 significantly decreased the mitochondrial membrane potential in isolated mitochondria. This detrimental effect on mitochondrial membrane integrity was attenuated when cofilin1 residues at position 139 and 147 were mutated to the nonoxidizable amino acid serine; and this effect was completely averted when all four cysteine residues were substituted by serine. These data suggest that oxidation of cofilin1 lead to a significant impairment of mitochondrial integrity and function. Accordingly, mitochondrial ROS accumulation was enhanced by the oxidized form of cofilin1 and, in line with the TMRE measurements, the 2Cys mutant did not exert mitochondrial ROS formation. These findings are in line with findings by Klamt et. al who evaluated the Cys139/147 mutant in a cellular environment by transfection of respective cofilin1-mutated plasmids [22].
Further, evaluation of the mitochondrial respiration revealed, that the wildtype form of cofilin1 impaired the oxygen consumption upon ADP injection, an indicator of complex II, III and Vdriven respiration. Although the mutation of Cys139 and 147 still led to a decrease of mitochondrial respiration, the effect was less pronounced compared to wildtype cofilin1.
In conclusion, the deleterious effect of cofilin1 was attenuated if either all cysteine residues of the protein were substituted by the non-oxidizable serine, or if cysteine residues at position 139 and 147 were mutated, indicating that both positions, Cys139 and Cys147, are crucial in mediating the direct damaging impact on mitochondria. However, the effects of the oxidized form of cofilin1 were always less pronounced compared to the positive controls (Antimycin A for mitochondrial-derived ROS and CCCP as an uncoupler to induce collapse of the mitochondrial membrane potential). A possible explanation is delivered by Liu and coworkers who demonstrated that cofilin1 needs the interaction with p53 for strong impacts on mitochondrial function [67]. Finally, our data demonstrate a striking effect of cofilin1 in cell death mechanisms linked to oxidative distress, underlining that cofilin1 acts as a redox sensor in cell death mechanisms comprising oxytosis and ferroptosis. However, it is tempting to speculate that thiol switches of cofilin1 generally have also regulatory functions in physiological signal transduction pathways. Thus, our data suggests, that interfering with cofilin1's activity by pharmacological inhibition or imposing cofilin1 phosphorylation at serine residue 3 could provide new potential therapeutic strategies for neurodegenerative diseases in the future.

. Cofilin1 is activated by dephosphorylation under conditions of oxidative stress
A HT22 cells were challenged with 10 mM glutamate for the indicated time and total as well as phosphorylated cofilin1 (Ser3) was analyzed via Western blot. Three blots were quantified (mean + SD). B Accordingly, HT22 cells were treated with 1 µM erastin for the specified time and total as well as phosphorylated cofilin1 (Ser3) was analyzed via Western blot and afterwards quantified from three independent blots. (mean + SD). Ctrl (control), pCof (phosphorylated cofilin1-ser3), Cof (cofilin1). ** p<0.01 compared to Ctrl (unpaired t-test).      were compared to the cofilin1 signal and to α-Tubulin as a loading control and presented as mean + SD. Ctrl (control); Glut (glutamate). B Primary cortical neurons from wildtype E18 pubs were exposed to the indicated concentration of CN03 3 h prior to 25 µM glutamate treatment for 24 h at DIV9. Data from n = 6 are shown as mean + SD. ### p<0.001 compared to control; *** p<0.001 compared to glutamate-treated control (ANOVA, Scheffé'stest). C Micromolar concentrations of glutamate stimulated excessive Ca 2+ entry into neurons, a pathologic condition known as excitotoxicity. By application of CN03 protein, a known Rhoactivator, cofilin1 is deactivated via ROCK-LIMK pathways thereby promoting neuronal protection by circumventing cofilin1 activation and mitochondrial demise. NMDA-R (N-methyl-D-aspartate receptor); ER (endoplasmic reticulum); [Ca 2+ ] (intracellular calcium concentration); P (Ser 3-phosphorylation); ROCK (Rho-associated serine/threonine kinase); LIMK (LIM kinase); DNA (deoxyribonucleic acid); roman numerals representing complex I-V of the respiratory chain.